Affinity binding complex formation is an essential step in biological or pharmaceutical phenomena. For example, the binding of proteins to DNA underlines many cellular activities including the control of gene expression, site-specific recombination, replication and repair of DNA damage. Enzyme-substrate interactions involve the recognition and binding of substrate by the enzyme as the first step. Hormones, neurotransmitters, lymphokines and other effector molecules bind to their receptors to initiate the cellular process which ultimately lead to achievement of their functions.
Consequently, affinity binding complexes are also important tools in biological or pharmaceutical research. For example, drug discovery often involves identification of binding factors of a particular target which mediates a disease. A variety of methods have been employed to detect affinity binding complex formation in order to identify the binding factors. For example, the gel electrophoresis mobility shift assay (EMSA) is the most commonly used method in the study of protein-DNA interactions. This method is based on the observation that binding of a protein to DNA fragments leads to a reduction in the electrophoretic mobility of the DNA fragment in non-denaturing polyacrylamide or agarose gels. While used extensively, EMSA requires relatively large amounts of sample and lengthy analysis time. Moreover, the assay is not suitable when dissociation of protein-DNA complex occurs during gel electrophoresis.
As another example, capillary electrophoresis (CE) combined with affinity recognition has gained a tremendous growth in recent years, with increasing biochemical, clinical, and pharmaceutical applications. A key element of the technique is the use of a molecular recognition agent, typically a protein that binds to a target molecule with high specificity and affinity. The complex formation can occur either before or during the electrophoretic separation, depending on the stability of the resultant complex. In applications such as CE-based immunoassays, however, tight binding of the analyte to the protein is essential to achieve a high degree of sensitivity and reproducibility. Ideally, the affinity complex thus formed should remain intact throughout the electrophoretic separation.
In current practice of affinity CE, the formation and stability of the complex are usually established by titration experiments, in which a series of solutions containing the substrate and its binding protein in various ratios are analyzed. The emergence of a new peak upon addition of the binding protein to the substrate is taken as the evidence for complex formation and the relative intensities corresponding to the complex and the free substrate are used for quantitation. The titration experiments have proved to be very useful in studies of binding interactions; however, they are time-consuming and unable to provide unequivocal identification of the complex when the complex is not well separated from the unbound molecules. Therefore, there remains a need for a simple and sensitive method to detect affinity complex formation.